The stimulation of the rate of the E. Coli membrane-bound transhydrogenase by ATP is mediated by the membrane-bound ATPase and the stimulation is abolished by inhibitors and uncouplers of oxidative phophorylation. The mechanism of energy coupling between these two enzymes is being studied with the purpose of gaining general information on energy transduction by membrane enzymes. The transhydrogenase, which has been purified, will be characterized as to molecular weight, amino acid composition, subunit structure, kinetic mechanism and substrate binding properties. The transhydrogenase will be incorporated into phospholipid vesicles to determine if it can generate a proton gradient, as measured by quenching of 9-amino-acridine fluoresence. The mechanism of repression of transhydrogenase by leucine will be determined and the assembly of the enzyme into the cell membrane following derepression will be studied. The orientation of the transhydrogenase in the cell membrane will be studied by comparing the effects of antibodies specific for each subnit on right side out and inverted membranes. Transhydrogenase mutants defective in energy coupling will be sought, by localized mutagenesis, for comparison with wild type enzyme.